Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ⌬Afatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ⌬Afatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ⌬Afatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ⌬Afatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.Nutrient limitation is one of the most significant stresses encountered by microorganisms in nature. Autophagy is a catabolic membrane trafficking system that counters such nutrient stress by initiating a process of limited intracellular digestion to support the organism during periods of reduced nutrient availability (26,32,37,60). The process begins with the formation of isolation membranes within the cytoplasm, whose origin is not entirely clear. These membranes progressively expand, nonselectively encapsulating cytosolic material into a doublemembrane vesicle called the autophagosome. The autophagosome fuses its outer membrane with a vacuole, releasing the autophagic body and its contents into the vacuolar lumen for degradation by resident hydrolases. Autophagy is upregulated in response to starvation stress, resulting in the generation of a pool of recycled molecules that provide the building blocks for continued synthesis of essential components until nutrient conditions improve (26,37,39,60). However, some autophagy remains constitutively active at low levels, even under nutrientreplete conditions, where it serves as a mechanism to remove old or damaged proteins and organelles (17,25,28,46,54). This provides an important form of quality control that combats the toxic accumulation of abnormal cytoplasmic components.The degradative functions of autophagy contribute to several important aspects of cell physiology, including autophagydependent programmed cell death (2, 14), cellular remodeling during development and differentiation (21,36,40,49,57), maintenance of endoplasmic reticulum homeostasis (4, 30, 6...

Aspergillus fumigatusCgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a ⌬cgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The ⌬cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37°C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the ⌬cgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37°C but had little effect on germination at room temperature. The ⌬cgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25°C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.Aspergillus fumigatus is a saprophytic filamentous fungus that inhabits soil, water, and organic debris, where it has an essential role in the recycling of carbon and nitrogen (37). The organism propagates itself by the release into the air of high concentrations of asexual spores (conidia), which are unavoidably inhaled on a daily basis (26,46). Since the conidia are efficiently cleared by normal defenses, their inhalation is of minor consequence to healthy individuals. However, in the absence of adequate host immunity, the conidia germinate into highly invasive hyphae that cause severe lung damage and eventually disseminate to other organs. Patients with depressed immunity are at increased risk for infection with A. fumigatus, and the prognosis for invasive disease is very poor in these individuals (40). Of particular concern is the rising incidence of aspergillosis, a situation that has arisen as a consequence of aggressive cancer treatments and the widespread use of potent immunosuppressive regimens that support organ transplantation (33,44,54,58).Since A. fumigatus conidia are no more prevalent in the environment than the spores of some nonpathogenic molds (34), it is generally assumed that the organism has unique features that allow it to survive in humans, and thermotolerance has long been suspected to play a role (37). As a major component of the biomass in a self-heating compost pile, A. fumigatus has evolved mechanisms that allow it to grow we...

SummaryWe have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DcasA/DcasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DcasA/DcasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DcasA/DcasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.